Nucleic acids and polypeptides specific of the Neisseria genus pathogenic strains

ABSTRACT

The invention concerns nucleic acids coding for polypeptides specific of the  Neisseria  genus pathogenic strains, the corresponding polypeptides, and their diagnostic and therapeutic applications.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/056,911, filed Mar. 27, 2008, now issued as U.S. Pat. No. 7,704,513,which is a divisional of U.S. application Ser. No. 10/909,436, filedAug. 3, 2004 now U.S. Pat. No. 7,384,768, which was a divisional of U.S.application Ser. No. 09/830,433, filed Aug. 16, 2001, now issued as U.S.Pat. No. 6,835,384, which is a National Stage of InternationalApplication No.: PCT/FR99/02643, filed Oct. 28, 1999, incorporatedherein by reference.

The present invention relates to nucleic acids encoding polypeptidesspecific for pathogenic strains of the Neisseria genus, in particularwhich are useful for preventing or treating a Neisseria meningitidisinfection.

In general, meningitis is either of viral origin or of bacterial origin.The bacteria mainly responsible are: type b Haemophilus influenzae,Neisseria meningitidis and Streptococcus pneumoniae. The Neisseriameningitidis species is subdivided into serogroups according to thenature of the capsular polysaccharides. Although about a dozenserogroups exist, 90% of meningitis cases can be attributed to threeserogroups: A, B and C.

Effective vaccines based on capsular polysaccharides exist forpreventing meningitis caused by Neisseria meningitidis serogroups A andC. These polysaccharides, unmodified, are only slightly immunogenic, ornot at all, in children under the age of two, and do not induce anyimmune memory. However, these drawbacks can be overcome by conjugatingthese polysaccharides to a carrier protein.

On the other hand, the polysaccharide of Neisseria meningitidisserogroup B is non-immunogenic, or relatively non-immunogenic in humans,whether or not it is in a conjugated form. Thus, it appears to be highlydesirable to seek a vaccine against meningitis caused by Neisseriameningitidis, in particular Neisseria meningitidis serogroup B, otherthan a vaccine based on polysaccharide.

To this end, various proteins of the external membrane of N.meningitidis have already been proposed, such as the membrane-boundreceptor for human transferrin (WO 90/12591 and WO 93/06861).

Neisseria meningitidis is genetically very close to Neisseriagonorrhoeae and Neisseria lactamica. N. gonorrhoeae is especiallyresponsible for infections located in the urogenital tract. It colonizesthe genital mucous membrane, crosses the epithelium and then invades thesub-epithelium, where it multiplies and is responsible for a severeinflammatory reaction. On the other hand, N. lactamica is considered tobe a nonpathogenic species.

Sequences present in N. gonorrhoeae and N. meningitidis, but absent fromN. lactamica, have been disclosed in patent application WO 98/02547, butthis prior patent application does not locate or identify the codingsequences.

The authors of the present invention have now managed to identify someof these genes by searching, in the meningococcal genome, for the openreading frames specific for pathogenic strains of the Neisseria genus,using the following strategy:

Some of the sequences disclosed in patent application WO 98/02547(referred to, in said prior application, as SEQ ID Nos 66, 67, 69, 70,72 to 96, 98 and 99) were positioned on the sequence of the genome ofthe N. meningitidis serogroup B strain (ATCC 13090), available from thePathoseq® bank of Incyte Pharmaceuticals, and also on the sequence ofthe genome of the Neisseria meningitidis strain Z2491 (Sanger Centre).This made it possible to identify, in the N. meningitidis genome whichhas 2.3 mega bases, 19 contigs representing 220 000 base pairs.

The authors of the present invention then analysed, for each of the 19contigs, the presence of open reading frames (ORFs) containing at least100 amino acids (and, by definition, bordered by an initiation codon anda stop codon), using the GENE JOCKEY® sequence processor program(Biosoft). This analysis made it possible to select approximately 400candidate ORFs.

The sequences of each of these ORFs were then analysed using the CODONUSE® program (Conrad Halling), which takes into account the codon usefrequency in N. meningitidis. Only the ORFs with sequences having amaximum frequency of use of these codons were selected. At the end ofthis analysis, 197 candidate ORFs were selected.

The ORFs selected using this double analysis were subjected to ahomology search through all of the available banks, using the BLASTX®program, from the access to the PATHOSEQ® bank of IncytePharmaceuticals. This homology search made it possible to exclude theORFs encoding, a priori, cytoplasmic or periplasmic proteins, inparticular metabolism proteins. The ORFs were also subjected to analysisof possible protein motifs, using the DNA Star Protean® program(Lasergene software).

The authors of the present invention then investigated whether the ORFsselected at the end of the previous step (118 in number) wereeffectively absent from N. lactamica, as predicted by the application ofthe prior art WO 98/02547.

To this end, a PCR amplification was carried out. This amplificationproved to be ineffective for 78 of the 118 ORFs tested. Only the ORFsfor which the amplification in N. lactamica was negative (sequencesnamed “lactamica ⁻”) were selected. In order to verify that thesenegative results were not “false negatives”, the lactamica ⁻ sequencesselected were subjected to a control by dot blot. At the end of thisstep, only 23 ORFs were confirmed N. meningitidis ⁺ /N. lactamica ⁻.

Finally, these 23 ORFs were repositioned in their entirety on the N.meningitidis ATCC13090 genome. This made it possible to demonstrate thatthree ORFs previously eliminated on the basis of their putative proteinfunction appeared to be located close to, or were even framed by, someof the 23 N. meningitidis ⁺ /N. lactamica ⁻ ORFs. These three ORFs (SEQID Nos 29, 35 and 37) were reintroduced into the study, and it wasproven that they were also N. meningitidis ⁺ /N. lactamica ⁻.

The authors of the present invention then attempted to discover whetherthe ORFs identified using the genome of the N. meningitidis serogroup Bstrain ATCC 13090 were also present in the genomes of N. meningitidisserogroup A 22491 (Sanger Centre) and of N. gonorrhoeae FA1090 (AdvancedCentre of Genome Technology, Oklahoma University). Then, they comparedthe sequences derived from these various genomes, with multiplealignment (Clustal, Infobiogen). This made it possible to redefine, forsome of the ORFs, the most probable position of the initiation codon andtranslation stop codon. The sequences of the open reading frames derivedfrom the strain ATCC13090 are given in the SEQ ID Nos 1-51 (odd numbers)and the amino acid sequences which are deduced therefrom are given inthe SEQ ID Nos 2-52 (even numbers).

A subject of the present invention is, therefore, a nucleic acid inisolated form encoding a polypeptide, or an antigenic fragment thereof,excluding the nucleic acids disclosed in SEQ ID Nos 70, 73, 74, 77, 80,81, 87, 88, 89, 94, 95 and 98 of application WO 98/02547 (sequencesattached to the present description and numbered SEQ ID Nos 130-141,respectively); said polypeptide having an amino acid sequence which isidentical or homologous to a sequence selected from those of group II;group II consisting of the sequences shown in SEQ ID Nos 2-52 (evennumbers) and the sequence SEQ ID No. 53.

Preferably, said nucleic acid can have a nucleotide sequence selectedfrom those of group I, group I consisting of the sequences shown in SEQID Nos 1-51 (odd numbers).

The term “nucleic acid” includes and means equally ORF, gene,polynucleotide, DNA and RNA. The term “nucleic acid in isolated form”means a nucleic acid separated from the biological environment in whichit is found under natural conditions. For example, a DNA molecule existsunder natural conditions when it is integrated into a genome or when itforms part of a library of genes. In that case, it cannot be in isolatedform. On the other hand, the same molecule separated from the genome bycloning (for example subsequent to a PCR amplification) should beconsidered as being in isolated form. Typically, a DNA molecule inisolated form does not contain the coding regions which are contiguouswith it in 5′ and 3′ in the genome from which it is derived. The nucleicacids in isolated form can be integrated into vectors (for exampleplasmids, or viral or bacterial vectors) without, even so, abandoningtheir characteristic of being separated from their natural environment.

The authors of the present invention have more particularly found thatthe ORFs which, when they are derived from the strain ATCC 13090, arecharacterized by the sequences as shown in SEQ ID Nos 19, 27, 39, 45,and 49 are specific for Neisseria meningitidis insofar as it has notbeen possible to demonstrate identical or homologous sequences in the N.gonorrhoeae genome. They have also found that the ORF characterized bythe strain sequence as shown in SEQ ID No. 39 is specific for Neisseriameningitidis serogroup B.

A subject of the invention is also a polypeptide in isolated form, or afragment thereof; said polypeptide having an amino acid sequenceidentical or homologous to a sequence selected from those of group II.

The amino acids framed in the sequence SEQ ID No. 8 correspond to thesignal sequence, and the amino acid in bold represents the first aminoacid of the mature form. The amino acid sequence of the mature proteinform is represented in SEQ ID No. 53.

In the context of the present invention, the terms “polypeptide” and“protein” are equivalent and mutually interchangeable. They refer to anyamino acid chain, whatever its length and its post-translationalmodifications (for example phosphorylation or glycosylation).

The expression “antigenic fragments of the polypeptides specific forpathogenic strains of the Neisseria genus” is intended to mean thepolypeptides derived from the polypeptides of the invention as definedabove, through deletions of portions of said polypeptides withoutdestroying the antigenicity (for example, without notable loss of theantigenic activity) of said polypeptides. The specific antigenicity canbe determined using various methods known to those skilled in the art,as explained later.

These fragments are preferably at least 12 amino acids long, morepreferably at least 20 amino acids long, preferentially 50 amino acidslong, more preferably still 75 amino acids long, preferentially 100amino acids long.

These fragments can be used to reveal epitopes which may be masked inthe parent polypeptides. They are also advantageous for inducing aT-lymphocyte-dependent protective immune response. The deletions can, infact, make it possible to eliminate immunodominant regions which arehighly variable between various strains.

Such fragments can be obtained using standard techniques known to thoseskilled in the art (for example, Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons Inc, 1994), for example by PCR,RT-PCR or treatment with restriction enzymes for the cloned DNAmolecules, or by the method of Kunkel et al. (Proc. Natl. Acad. Sci. USA(1985) 82:448).

The expression “homologous amino acid sequence” is intended to mean asequence which differs from one of the sequences of group II bysubstitution, deletion and/or insertion of one or more amino acids, atpositions such that these modifications do not destroy the specificantigenicity of the polypeptide in question.

Said substitutions are preferably conservative substitutions, i.e.substitutions of amino acids of the same class, such as substitutions ofamino acids with uncharged side chains (for instance asparagine,glutamine, serine, threonine and tyrosine), of amino acids with basicside chains (for instance lysine, arginine and histidine), of aminoacids with acid side chains (for instance aspartic acid and glutamicacid) or of amino acids with apolar side chains (for instance glycine,alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan and cysteine).

Advantageously, a homologous amino acid sequence has at least a 75%degree of homology (i.e. of identity) with one of the sequences of groupII; preferably this degree of homology is at least 80%, most preferablyat least 90%. The homologous amino acid sequences include, inparticular, the sequences which are substantially identical to one ofthe sequences of group II. The expression “substantially identicalsequence” means a sequence which has at least a 90%, advantageously atleast a 95%, preferably at least a 97%, and most preferably at least a99%, degree of homology (i.e. of identity) with one of the sequences ofgroup II. In addition, it may differ from the reference sequence onlythrough mainly conservative substitutions.

The degree of homology (also named degree of identity) is generallydetermined using a sequence analysis program (for example, SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Centre, 1710 University Avenue, Madison, Wis.53705). Similar amino acid sequences are aligned so as to obtain themaximum degree of homology (i.e. identity). To this end, it may benecessary to artificially introduce gaps into the sequence. Once optimalalignment has been produced, the degree of homology (i.e. identity) isestablished by recording all the positions for which the amino acids ofthe two sequences compared are identical, with respect to the totalnumber of positions.

The expression “homologous nucleotide sequences” is intended to meansequences which differ from the sequences of group I by substitution ofone or more nucleotides, or by deletion and/or insertion of one or morecodons, at positions such that these sequences (i) still encodepolypeptides having the sequences of group II, due to the effect of thedegeneracy of the genetic code; or (ii) encode polypeptides havinghomologous sequences as defined above.

Advantageously, a homologous nucleotide sequence has at least a 60%degree of homology with one of the sequences of group I; preferably thisdegree of homology is at least 80%, most preferably at least 90%.

Typically, a homologous nucleotide sequence hybridizes specifically tothe sequences complementary to the sequences of group I, under stringentconditions. The temperature at which the hybridization assay is carriedout constitutes an important factor which influences the stringency.Conventionally, this temperature, termed hybridization temperature (Th),is selected from 5 to 40° C., preferably from 20 to 25° C., below thetemperature at which 50% of the paired strands separate (Tm). Ingeneral, it is considered that conditions of high stringency aresatisfied when Th is lower than Tm by 5 to 25° C. approximately, forexample by 5 to 10° C. or, most commonly, by 20 to 25° C. approximately.Moderate stringency is established when Th is lower than Tm by 30 to 40°C.

For sequences comprising more than 30 bases, the temperature Tm isdefined by the equation: Tm=81.5+0.41(% G+C)+16.6 Log(cationconcentration)−0.63(% formamide)−(600/number of bases). Thus, ionicstrength has a major impact on the value of Tm. The temperature Tmincreases by 16.6° C. every time the monovalent cation concentrationincreases by a factor of 10. The addition of formamide into thehybridization buffer causes, on the other hand, the value of Tm todecrease. (For a complete reference, see Sambrook et al., MolecularCloning, A laboratory manual, Cold Spring Harbor Laboratory Press, 1989,pages 9.54-9.62).

Conventionally, hybridization experiments are carried out at atemperature of 60 to 68° C., for example at 65° C. At this temperature,stringent hybridization conditions can, for example, be implemented in6×SSC, advantageously in 2×SSC or 1×SSC, preferably in 0.5×SSC, 0.3×SSCor 0.1×SSC (in the absence of formamide). A solution of 1×SSC contains0.15 M of NaCl and 0.015 M of sodium citrate.

For this reason, in other words, a subject of the invention is apolynucleotide in isolated form, which is capable of hybridizing, understringent conditions, with a DNA molecule having one of the nucleotidesequences as shown in SEQ ID Nos 1-51 (odd numbers) or the sequencescomplementary thereto.

A specific class of homologous sequences consists of those encounterednaturally by virtue of the extremely common phenomenon of allelicvariation. A bacterial species, for example N. meningitidis or N.gonorrhoeae, consists of a large variety of strains which differ fromone another through minor variations, termed allelic variations. Thus, apolypeptide which is present in various strains and which, of course,performs the same biological function in each of them, can have an aminoacid sequence which is not identical from one strain to the other. Inother words, the sequences derived from the allelic variation are purelysequences equivalent or alternative to those of group II. The class ofsequences which are allelic variants of one of the sequences of group IIconsists of the sequences of the polypeptide as found in a pathogenicspecies of the Neisseria genus (for example, N. meningitidis or N.gonorrhoeae) other than the N. meningitidis strain ATCC 13090. Thebiological function which is associated with the allelic variantsequences is the same as that which is associated with the referencesequence. The differences (substitution, deletion or addition of one ormore amino acids) which they exhibit between one another (including thereference sequence) do not modify the biological function of thepolypeptide. The term “biological function” is intended to mean thefunction exercised by the polypeptide in the cells which produce itnaturally.

The allelic variation is also expressed in the coding sequences. Apolynucleotide, encoding a polypeptide, having a sequence which is anallelic variant of one of the sequences of group I can be easily clonedby amplifying the genomic DNA of the strains of pathogenic species ofthe Neisseria genus, for example by PCR (polymerase chain reaction),using synthetic oligonucleotide primers capable of hybridizing to the 5′and 3′ ends of the coding region. The sequences of such primers caneasily be established by those skilled in the art using the nucleotidesequences given in SEQ ID Nos 1-51 (odd numbers). The primers generallyhave from 10 to 40 nucleotides, preferably from 15 to 25 nucleotides.

For this reason, in other words, a subject of the invention is a DNAmolecule in isolated form which can be amplified and/or cloned by PCRfrom the genome of a pathogenic Neisseria strain, using a pair of 5′ and3′ PCR primers; the sequences of these primers being established usingone of the nucleotide sequences as shown in SEQ ID Nos 1-51 (oddnumbers). An example is given, for each pair of primers, in Example I.1hereinafter.

A subject of the present invention is more particularly the allelicvariants having the nucleotide sequences SEQ ID Nos 54 to 76 (evennumbers) and the products encoded by these nucleotide sequences, havingthe amino acid sequences SEQ ID Nos 55 to 77 (odd numbers).

The polypeptides of the invention can be fused to other polypeptides,for example by translation of a hybrid gene. Vectors for expressingfusion polypeptides are commercially available, such as the vectorspMal-c2 or pMal-p2 from New England Biolabs, in which the protein towhich the polypeptides of the invention can be fused is amaltose-binding protein, the glutathione-S-transferase system fromPharmacia or the His-Tag system from Novagen. Such systems are inparticular useful for purifying the polypeptides of the invention. Thepolypeptides of the invention can be fused to polypeptides havingadjuvant activity, such as for example the B subunit of cholera toxin orthe B subunit of the E. coli heat-sensitive toxin.

The nucleic acids of the present invention can be used (i) in a processfor producing the polypeptides encoded by said nucleic acids, in arecombinant host system, (ii) for the construction of vaccinationvectors, such as poxviruses, intended to be used in methods andcompositions for preventing and/or for treating an infection withpathogenic Neisseria strains, in particular with Neisseria meningitidis,(iii) as a vaccination agent in a naked form or in combination with avehicle which promotes transfer to the target cells and, (iv) in theconstruction of attenuated Neisseria strains which can overexpress anucleic acid of the invention, or express it in a non-toxic, mutatedform.

The present invention also provides (i) an expression cassettecontaining a polynucleotide of the invention placed under the control ofelements allowing its expression, in particular under the control of asuitable promoter; (ii) an expression vector containing said expressioncassette; (iii) a host cell (prokaryotic or eukaryotic) transformed withan expression cassette and/or an expression vector as defined above, and(iv) a method for obtaining a polypeptide encoded by said polynucleotideof the invention, comprising culturing said transformed cell underconditions allowing the expression of the polynucleotide of theinvention, and recovering the polypeptide from the cell culture.

Among the eukaryotic hosts which can be used, mention may be made inparticular of yeast cells (for example Saccharomyces cerevisiae orPichia Pastoris), mammalian cells (for example COS 1, NIH3T3 or JEG3)arthropod cells (for example Spodoptera frugiperda (SF9)) and plantcells. Among the prokaryotic hosts which can be used, mention may bemade in particular of E. coli.

The choice of the expression cassette depends on the host system chosen,and also on the characteristics desired for the expressed polypeptide.In general, expression cassettes include a promoter which is functionalin the host system selected and which can be constitutive or inducible;a ribosome-binding site; an initiation codon (ATG); if necessary, aregion encoding a signal peptide; a nucleotide sequence of theinvention; a stop codon; and, optionally, a 3′ terminal region(translation and/or transcription terminator). The open reading frame(ORF) consisting of the nucleotide sequence of the invention, alone orassociated with the region encoding the signal peptide, is placed underthe control of the promoter such that translation and transcription takeplace in the host system. The promoters and regions encoding the signalpeptides are known to those skilled in the art. Among them, mention maybe made in particular of the arabinose-inducible promoter (araBpromoter) of Salmonella typhimurium, which is functional in Gram⁻bacteria such as E. coli (U.S. Pat. No. 5,028,530 and Cagnon et al.,Protein Engineering (1991) 4(7): 843), the promoter of the T7bacteriophage gene encoding RNA polymerase (U.S. Pat. No. 4,952,496),and the OspA and RlpB signal peptide (Takase et al., J. Bact. (1987)169:5692).

The polypeptide expressed can be recovered in a practically purifiedform from the cell extract or from the supernatant, after centrifugingthe recombinant cell culture. The recombinant polypeptide can, inparticular, be purified using methods of affinity purification with theaid of antibodies, or using any other method known to those skilled inthe art, for instance by genetic fusion with a small binding domain.

The nucleic acids of the invention can also be used in the field ofvaccination, either by using a viral or bacterial host as a vehicle forreleasing the DNA, or by administering the nucleic acid of interest in afree form.

A subject of the present invention is also (i) a vaccination vectorcontaining a nucleic acid of the invention, placed under the control ofelements allowing its expression; (ii) a pharmaceutical compositioncontaining a therapeutically or prophylactically effective amount ofsaid vaccination vector; (iii) a method for inducing an immune responseagainst Neisseria in a vertebrate, in particular a mammal, preferably ahuman, said method comprising the administration to said vertebrate ofan immunologically effective amount of said vaccination vector so as tocause an immune response, in particular a protective or therapeuticresponse to Neisseria meningitidis; and (iv) a method for preventingand/or treating an infection with pathogenic Neisseria strains, inparticular with Neisseria meningitidis, which comprises theadministration of a prophylactic or therapeutic amount of saidvaccination vector of the invention to an individual requiring such atreatment.

In combination with the polypeptides of the invention, the vaccinationvector as defined above can also comprise nucleotide sequences theexpression of which allows the immune response to be stimulated, such asthe sequences encoding cytokines.

Said vaccination vector of the invention can be administered via anyroute which is conventional in the field of vaccination, in particularvia the parenteral route (for example subcutaneous, intradermal,intramuscular, intravenous or intraperitoneal route). The dose dependson many parameters which are known to those skilled in the art, such asthe vector itself, the route of administration, or the weight, age orsex of the animal or of the human to be vaccinated.

A subject of the present invention is also (i) a pharmaceuticalcomposition containing a therapeutically or prophylactically effectiveamount of a polynucleotide of the invention; (ii) a method for inducingan immune response against pathogenic Neisseria strains, in particularNeisseria meningitidis in a vertebrate, by administering to saidvertebrate an immunologically effective amount of said polynucleotide soas to cause an immune response, in particular a protective immuneresponse against pathogenic Neisseria strains, especially Neisseriameningitidis; and (iii) a method for preventing and for treating aninfection with pathogenic Neisseria strains, in particular withNeisseria meningitidis, by administering a therapeutic or prophylacticamount of said polynucleotide to an individual requiring such atreatment.

The polynucleotides of the invention (DNA or RNA) can be administered toa vertebrate as they are. When a DNA molecule of the invention is used,it can be in the form of a plasmid incapable of replicating in avertebrate cell and incapable of integrating the genome of saidvertebrate. Said DNA molecule is, typically, placed under the control ofa promoter suitable for expression in a vertebrate cell. Saidpolynucleotide used as vaccine can be formulated according to variousmethods known to those skilled in the art. Said polynucleotide can, inparticular, be used in a naked form, free of any vehicle which promotestransfer to the target cell, such as anionic liposomes, cationic lipids,microparticles, for example gold microparticles, precipitation agents,for example calcium phosphate, or any other agent which facilitatestransfection. In this case, the polynucleotide can be simply diluted ina physiologically acceptable solution, such as a sterile solution or asterile buffer solution, in the presence or absence of a vehicle. Whenit is present, this vehicle can be preferably isotonic, hypotonic orslightly hypertonic, and has a relatively low ionic strength. It can,for example, be a sucrose solution (for example a solution containing20% of sucrose).

Alternatively, a polynucleotide of the invention can be combined withagents which facilitate transfection. It can be, inter alia, (i)combined with a chemical agent which modifies cell permeability, such asbupivacaine (WO 94/16737); (ii) encapsulated in liposomes, optionally inthe presence of additional substances which facilitate transfection (WO93/18759, WO 93/19768, WO 94/25608 and WO 95/2397, WO 93/18759 and WO93/19768); or (iii) combined with cationic lipids, or silica, gold ortungsten microparticles.

When the polynucleotides of the invention coat microparticles, theseparticles can be injected via the intradermal or intraepidermal route,using the “gene gun” technique (U.S. Pat. No. 4,945,050, U.S. Pat. No.5,015,580 and WO 94/24263).

The amount of DNA to be used as a vaccine depends, in particular, on thestrength of the promoter used in the DNA construct, on theimmunogenicity of the product expressed, on the individual to which thisDNA is administered, on the method of administration and on the type offormulation. In general, a therapeutically or prophylactically effectiveamount ranging from approximately 1 μg to approximately 1 mg, preferablyfrom approximately 10 μg to approximately 800 μg, and preferentiallyfrom approximately 25 μg to approximately 250 μg, can be administered tohuman adults.

The polynucleotide of the invention can be administered via anyconventional route of administration, such as in particular via theparenteral route. The choice of the route of administration depends, inparticular, on the formulation chosen. A polynucleotide formulated incombination with bupivacaine is advantageously administered into muscle.When neutral or anionic liposomes, or a cationic lipid such as DOTMA(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) orDC-Chol (3-beta-(N—(N′,N′-dimethyl-aminomethane)carbamoyl)cholesterol)are used, the formulation can advantageously be injected via theintravenous, intramuscular, intradermal or subcutaneous route. Apolynucleotide in a naked form can advantageously be administered viathe intramuscular, intradermal or subcutaneous route.

The nucleotide sequences of the invention allow the construction ofspecific nucleotide probes and primers which can be used in diagnosis.Said probes or primers are nucleic acids having sequences identical orhomologous to portions of the sequences of group I or to the sequencescomplementary thereto.

Preferably, said probes contain from approximately 5 to approximately100, preferably from approximately 10 to approximately 80, nucleotides.They can contain modified bases, the sugar and phosphate residuespossibly also being modified or substituted. The probes of the inventioncan be used in diagnostic tests, to capture or detect polynucleotidesspecific for pathogenic Neisseria strains. Such capture probes canconventionally be immobilized on a solid support directly or indirectly,by covalent bonding or by passive adsorption. A detection probe can belabelled, in particular with a radioactive isotope, an enzyme such asperoxidase or alkaline phosphatase, or enzymes capable of hydrolyzing achromogenic, fluorogenic or luminescent substrate, or with compoundswhich are, themselves, chromogenic, fluorogenic or luminescent,nucleotide analogues; or biotin.

A primer generally contains from approximately 10 to approximately 40nucleotides, and can be used to initiate enzymatic polymerization of theDNA in an amplification process (for example PCR), in an elongationprocess or in a reverse transcription method. A primer of the inventioncan in particular be a primer as described in Example II.1 hereinafter.

A subject of the present invention is also:

-   -   (i) a reagent containing a probe of the invention for detecting        and/or identifying the presence of pathogenic Neisseria strains        in a biological sample;    -   (ii) a process for detecting and/or for identifying the presence        of pathogenic Neisseria strains in a biological sample, said        method comprising the steps consisting in a) extracting the DNA        or RNA from a biological sample and denaturing it; b) exposing        said DNA or said RNA to a probe of the invention, under        stringent hybridization conditions, so as to detect the        hybridization; and    -   (iii) a method for detecting and/or for identifying pathogenic        Neisseria strains in a biological sample, in which the DNA is        extracted from a biological sample and mixed together with at        least one and preferably with two primers of the invention, and        is amplified, for example by PCR.

As mentioned above, the polypeptides produced by the expression of theORF sequences identified can be used as vaccination agents. The specificantigenicity of the polypeptides homologous to the polypeptides havingsequences of group II can be evaluated by assaying the cross-reactivitywith an antiserum directed against the polypeptides having sequences ofgroup II. A monospecific hyperimmune antiserum can be produced against apurified polypeptide having a sequence of group II or a fusionpolypeptide, for example an expression product of the MBP, GST orHis-tag systems.

The specific antigenicity can be determined using various methods knownto those skilled in the art, in particular the Western blot, dot blotand ELISA techniques, described below.

In the Western blot technique, the protein preparation to be tested issubjected to SDS-PAGE gel electrophoresis. After transfer onto anitrocellulose membrane, the material is incubated with a monospecifichyperimmune antiserum obtained after having immunized an animal with thereferent material; i.e., in the present case, with a polypeptide havingan amino acid sequence of group II. This antiserum is diluted beforehandin a dilution range of approximately 1:50 to 1:5000, preferably ofapproximately 1:100 to 1:500. The specific antigenicity is revealed whena band corresponding to the product shows reactivity with one of thedilutions above.

In the ELISA assay, a purified protein preparation is preferably used,although a whole cell extract may also be used. Approximately 100 μl ofa preparation at approximately 10 μg/ml are distributed into the wellsof a plate. The plate is incubated for two hours at 37° C., and thenovernight at 4° C. The plate is then washed with a phosphate bufferedsaline solution (PBS) comprising 0.05% of Tween 20. The wells aresaturated with 250 μl of PBS containing 1% of bovine serum albumin (BSA)so as to prevent non-specific antibody binding. After incubation for onehour at 37° C., the plate is washed with the PBS/Tween buffer. Theantiserum is serially diluted in PBS/Tween buffer containing 0.5% BSA.100 μl of this dilution are added per well. The plate is incubated for90 minutes at 37° C., washed and evaluated according to standardprocedures. For example, when specific antibodies are produced inrabbits, a goat anti-rabbit peroxidase conjugate is added to the wells.The incubation is carried out for 90 minutes at 37° C. and the plate isthen washed. The reaction is measured by colorimetry (the reaction ispositive when the optical density value is 1, if the dilution is atleast 1:50, preferably at least 1:500).

In the dot blot assay, a purified protein is preferably used, it beingunderstood that it is also possible to use a whole cell extract.Two-fold serial dilutions of a protein solution at approximately 100μg/ml are prepared in a 50 mM Tris-HCl buffer, pH: 7.5. 100 μl of eachdilution are applied to a nitrocellulose membrane (BioRad apparatus).The buffer is removed by applying suction to the system. The wells arewashed by adding 50 mM of Tris-HCl buffer (pH: 7.5) and the membrane isair-dried. The membrane is then saturated in a blocking buffer (50 mMTris-HCl (pH: 7.5) 0.15 M NaCl, 10 g/l of skimmed milk) and incubatedwith a dilution of antiserum ranging from approximately 1:50 to 1:5000,preferably to approximately 1:500. The reaction is revealed inaccordance with standard procedures. For example, when specificantibodies are produced in rabbits, a goat anti-rabbit peroxidaseconjugate is added to the wells. The incubation is carried out for 90minutes at 37° C. The reaction is developed with the suitable substrateand measured, for example by colorimetry, by the appearance of acoloured spot (a reaction is positive when a coloured spot appears inassociation with a dilution of at least 1:50, preferably of at least1:500).

A subject of the present invention is also (i) a pharmaceuticalcomposition containing a therapeutically or prophylactically effectiveamount of a polypeptide of the invention; (ii) a method for inducing animmune response against pathogenic Neisseria strains in a vertebrate, byadministering to said vertebrate an immunogenically effective amount ofa polypeptide of the invention so as to cause an immune response, inparticular a protective immune response against pathogenic Neisseriastrains; and (iii) a method for preventing and/or for treating aninfection with pathogenic Neisseria strains, by administering atherapeutic or prophylactic amount of a polypeptide of the invention toan individual requiring such a treatment.

The immunogenic compositions of the invention can be administered viaany route which is conventional in the field of vaccination, inparticular via the parenteral route (for example subcutaneous,intradermal, intramuscular, intravenous or intra-peritoneal route). Thechoice of the route of administration depends on a certain number ofparameters, such as the adjuvant combined with the polypeptide.

A composition of the invention contains at least one polypeptide asdefined above. It can also contain at least one additional antigen ofNeisseria meningitidis and/or Neisseria gonorrhoeae.

The polypeptides of the invention can be formulated with liposomes,preferably neutral or anionic liposomes, microspheres, ISCOMS or“virus-like” particles, in order to facilitate the transfer of thepolypeptide and/or to increase the immune response.

The administration can be carried out with a single dose or with dosesrepeated, if necessary, at intervals which can be determined by thoseskilled in the art.

For example, an initial dose can be followed by three booster doses atintervals of one or more weeks or of one or more months. The suitabledose depends on many parameters, including the individual treated (adultor child), the specific vaccination antigen, the route of administrationand the frequency of administration, the presence or absence or the typeof adjuvant, and the desired effect (for example protection and/ortreatment), and can be determined by those skilled in the art. If theroute of administration is parenteral, the dose is preferentially lessthan 1 mg, preferably approximately 100 μg. The polypeptides andpolynucleotides of the invention used as vaccination agents can be usedsequentially, in a several-step immunization process. For example, avertebrate can be initially sensitized with a vaccination vector of theinvention, such as a poxvirus, for example via the parenteral route, andcan then be stimulated twice with the polypeptide encoded by thevaccination vector.

A polypeptide of the invention can also be useful as a diagnostic agentfor detecting the presence of anti-Neisseria meningitidis and/oranti-Neisseria gonorrhoeae antibodies in a biological sample such as ablood sample.

A subject of the present invention is also monospecific antibodiesdirected against the polypeptides of the invention.

The term “monospecific antibodies” is intended to mean an antibodycapable of reacting specifically with a Neisseria polypeptide of theinvention. Such antibodies can be polyclonal or monoclonal, and can berecombinant antibodies, for example chimeric (for example consisting ofa variable region of murine origin associated with a constant region ofhuman origin), humanized and/or single-chain antibodies. Said antibodiescan also be in the form of immunoglobulin fragments, for example F(ab)′2or Fab fragments. The antibodies of the invention can be of any isotype,for example IgA or IgG, the polyclonal antibodies possibly being of asingle isotype or possibly containing a mixture of several isotypes.

The antibodies of the invention directed against the polypeptides of theinvention can be produced and identified using standard immunologicalmethods, for example Western blot analysis, a dot blot assay, an ELISAassay (Coligan et al., Current Protocols in Immunology (1994) John Wiley& Sons, Inc., New York, N.Y.). Said antibodies can be used in diagnosticprocesses for detecting the presence of a Neisseria meningitidis antigenin a sample such as, in particular, a biological sample (for example ablood sample).

The antibodies of the invention can also be used in affinitychromatography processes for purifying a polypeptide of the invention.Finally, such antibodies can also be used in prophylactic or therapeuticpassive immunization methods.

A subject of the present invention is also a diagnostic method fordetecting the presence of pathogenic Neisseria strains in a biologicalsample, comprising bringing said biological sample into contact with anantibody or a polypeptide of the invention, such that an immune complexis formed, and detecting said complex which indicates pathogenicNeisseria strains in the organism from which the sample originates.Those skilled in the art understand that the immune complex is formedbetween a component of the sample and the antibody or the polypeptide ofthe invention, any substance not bound possibly being eliminated priorto the detection of the complex.

Thus, a reagent of polypeptide type can be used for detecting thepresence of anti-Neisseria meningitidis and/or Neisseria gonorrhoeaeantibodies in a sample, whereas an antibody of the invention can be usedas a reagent for assaying the presence of a Neisseria meningitidisand/or Neisseria gonorrhoeae polypeptide in a sample.

For use in diagnostic applications, the reagent (for example theantibody or the polypeptide of the invention) can be in the free stateor immobilized on a solid support, by direct or indirect means.

The direct means include passive adsorption or covalent bonding betweenthe support and the reagent.

The term “indirect means” is intended to mean that a substance whichinteracts with said reagent is attached to the solid support. Forexample, if a reagent of polypeptide type is used, an antibody whichbinds to this polypeptide can be used as an anti-reagent substance, itbeing understood that this substance binds to an antibody which is notinvolved in recognizing the antibodies in the biological samples.

Among the indirect means which can be used, mention may also be made ofthe ligand receptor system, a molecule such as a vitamin possibly beinggrafted onto the reagent of polypeptide type, and the correspondingreceptor possibly being immobilized on the solid phase. This isillustrated by the biotin-streptavidin system. It is also possible toadd a peptide tail to the reagent, by chemical engineering or geneticengineering, and to immobilize the grafted or fused product by passiveadsorption or covalent bonding with the peptide tail.

A subject of the present invention is also a process for purifying, froma biological sample, a Neisseria polypeptide of the invention, byaffinity chromatography with a monospecific antibody of the invention.Said antibody is preferably of isotype IgG.

According to an example of implementation, a biological sample,preferably in a buffer solution, is applied to a chromatographicmaterial, preferably equilibrated with the buffer used to dilute thebiological sample, such that the polypeptide of the invention (i.e. theantigen) may adsorb to the material. The unbound components are washedand the antigen is then eluted with a suitable elution buffer, such as aglycine buffer or a buffer containing chaotropic agent, for exampleguanidine HCl, or a high concentration of salt (for example 3M MgCl₂).The eluted fractions are recovered and the presence of antigen isdetected, for example by measuring the absorbence at 280 nm.

A subject of the present invention is also (i) a pharmaceuticalcomposition containing a therapeutically or prophylactically effectiveamount of a monospecific antibody of the invention; and (ii) a methodfor preventing and/or for treating an infection with pathogenicNeisseria strains, by administering a therapeutic or prophylactic amountof a monospecific antibody of the invention to an individual requiringsuch a treatment.

To this end, the monospecific antibody of the invention is preferably ofisotope IgG, and preferably fixes the complement. Said monospecificantibody according to the invention can be administered alone or in amixture with at least one other monospecific antibody, specific for adifferent Neisseria meningitidis and/or Neisseria gonorrhoeaepolypeptide, according to the invention. The amount of antibody can bedetermined easily by those skilled in the art. For example, a dailyadministration of approximately 100 to 1000 mg of antibodies over aweek, or three daily doses of approximately 100 to 1000 mg of antibodiesover two or three days, may be an effective dose.

The therapeutic or prophylactic effectiveness may be evaluated usingstandard methods known to those skilled in the art, for example bymeasuring the induction of an immune response or the induction ofprotective and/or therapeutic immunity (in newborn rats or mice),through evaluation of the bacterial load in the cerebrospinal fluid. Theprotection can be determined by comparing the degree of Neisseriainfection to a control group. Protection is demonstrated when theinfection is decreased in comparison with the control group. Such anevaluation can be carried out with the polynucleotides, the vaccinationvectors, the polypeptides and also the antibodies according to theinvention. The therapeutic or prophylactic effectiveness of a productaccording to the invention (polynucleotide or polypeptide) can also beevaluated in an assay for bactericidal activity, as described by Danveet al., Vaccine (1993) 11 (12):1214 against the meningococcal strain oforigin of the polynucleotide or polypeptide used. In the field ofmeningococcal vaccines, the bactericidal activity assay is, in fact,recognized as being the reference assay based upon which it is possibleto make a valid prediction of the vaccination value of a product.Briefly, a product according to the invention is administered to animalssuch as rabbits in order to produce an antiserum against this product.Then, this antiserum is assayed for its lysis capacity. The bactericidaltitre of an antiserum represents the inverse of the dilution of thisantiserum for which 50% of the load of meningococci is lysed. Theantiserum is considered to be bactericidal when the titre is higher than4, with respect to the menigococcal strain of origin of thepolynucleotide or polypeptide used. In that case, the product againstwhich the antiserum was generated is demonstrated to be potentiallyadvantageous from a pharmaceutical point of view.

The following examples illustrate the invention without limiting thescope thereof.

LEGEND OF THE FIGURE

The attached FIGURE represents the vector pCAMyc-His used as a cloningvector.

DETAILS OF THE STRATEGY FOR IDENTIFYING THE ORFS

In order to select the ORF sequences specific for the pathogenic strainsof the Neisseria genus, a PCR amplification is carried out on thesequences of the 118 ORFs selected after analysis with the Gene Jockey®,Codon Use®, and homology search programs. Only the sequences for whichthe amplification in N. lactamica is negative (sequences named“lactamica ⁻”) are selected. In order to verify that these negativeresults are not “false negatives”, the lactamica ⁻ sequences selectedare subjected to a dot blot.

A—PCR Amplification:

A.1. Extraction of Genomic DNAs:

The genomic DNAs of all of the Neisseria strains used in this study wereprepared according to an identical protocol. The N. meningitidis, N.lactamica, N. flava, N. subflava and N. mucosa strains were cultured ontissues of MHA (Muller Hinton Agar, Difco) medium, and the N.gonorrhoeae were cultured on tissues of MHA medium supplemented with 10%of heat-treated horse blood (Biomérieux) and 1% of Isovitalex(Biomérieux). The culturing is carried out under an atmospherecontaining 10% CO₂, overnight at 37° C. Then, the cells are harvested,and washed in PBS phosphate buffer (pH 7.2), and the DNA is extractedaccording to protocol D of the “Rapid Prep genomic DNA isolation kit forcells and tissue” (Pharmacia Biotech).

The genomic DNAs were then controlled on agarose gel for theircompleteness and by PCR reaction for their purity.

A.2. PCR Reaction for Screening the ORFs Absent in N. lactamica 2314:

A PCR amplification was carried out on the genomic DNAs of the N.meningitidis strain ATCC 13090 and N. lactamica strain 2314 (ATCC23970), according to the following protocol:

The PCR reaction was carried out on a 50 μl volume with 10 ng of genomicDNA, 250 μM of each of the dNTPs, 300 nM of each of the primers, 1× TaqDNA polymerase buffer and 2 u of Taq DNA polymerase (Appligène).

The amplification cycles are:

97° C.   45 seconds 25 cycles 56° C.   1 minute 25 cycles 72° C. 2.30minutes 25 cycles

For each of the ORFs analyzed, positive and negative controls for thePCR reaction were carried out. At this stage, only the N. meningitidis+and N. lactamica− ORFs are selected.

B—Selection of the N. meningitidis ⁺ N. lactamica ⁻ ORFs by Dot Blot onGenomic DNA:

The absence of a product of PCR amplification of an ORF with genomic DNAof N. lactamica 2314 as the matrix does not guarantee the absence ofthis ORF in the N. lactamica 2314 genome. Specifically, a certainvariability in the region to which the oligonucleotides should hybridizemay be responsible for the absence of amplified product for a given ORF.

In this context, further verification is carried out by dot blot ongenomic DNA, using, as probe, the products of genomic amplification onthe N. meningitidis strain corresponding to each of the reading framesidentified. The dot blot filters contain genomic DNA of the followingstrains: 2 N. lactamica strains 8064 and 2314, one N. flava strain ATCC30008, one N. mucosa strain ATCC 9297, 3 N. meningitidis serogroup Bstrains ATCC13090, M982 and B16B6, one N. meningitidis serogroup Astrain 22491, one N. meningitidis serogroup C strain (strain 24182) and2 N. gonorrhoeae strains MS11 and FA1090. This dot blot analysis makesit possible to validate the absence of the ORF in N. lactamica 2314 and8064, and it is also an indication of the degree of variability of anORF within the Neisseria strains.

The dot blot technique used is as follows. Approximately 50 ng ofgenomic DNA, denatured for 5 min at 100° C., of the various Neisseriastrains are loaded, with suction, onto a Hybond N+ nitrocellulosemembrane (Amersham) placed between the jaws of a dot blot apparatus(BioRad). Then, the DNA is fixed on the membranes for 5 min with UVradiation at 315 nm.

The membranes are incubated in a prehybridization buffer (containingdenatured salmon sperm DNA). They are then hybridized with a probecorresponding to the product of amplification of the ORF of interest,labelled according to a cold labelling protocol, such as the “DIG DNAlabelling and detection kit” system (Boehringer Mannheim).

The ORF which does not hybridize to the genomic DNA of N. lactamica 2314and 8064 is definitively selected as a potential vaccination candidate.

EXAMPLE I Cloning

1. PCR Amplification

Each of the ORFs was amplified by PCR using the genomic DNA of N.meningitidis serogroup B (strain ATCC 13090), according to standardprotocol.

Two oligonucleotides, primers on the N-terminal side and on theC-terminal side were defined for each of the ORF sequences of theinvention.

The primer on the N-terminal side comprises an enzyme restriction sitefor cloning, a CCACC Kozak sequence for translation initiation (M.Kozak, J. Mol. Biol. 196: 947-950), the ATG of the potential ORF andapproximately 17 bases specific for the 5′ portion of the ORF.

The primer on the C-terminal side was defined such that the ORF clonedis in fusion, in its 3′ portion, with a repeat of 8 histidines and astop codon which are present in the vector behind the multiple cloningsite, hence the insertion of an “A” base in order to keep the correctreading frame after cloning and the disappearance of the stop codon ofthe ORF. The primer on the C-terminal side thus comprises an enzymerestriction site for cloning, an “A” base, and then approximately 20bases specific for the 3′ portion of the gene starting from the codonpreceding the stop codon.

After searching for restriction sites which are absent in each of theORFs, with the aid of the DNASTAR MapDraw subprogram (LasergeneSoftware), the XbaI restriction site in 5′ and BglII restriction site in3′ are used for the ORF SEQ ID No. 19. For the ORF SEQ ID No. 41, theSpeI site in 5′ and the BglII site in 3′ are used. The XbaI restrictionsite in 5′ and BamHI restriction site in 3′ are used to clone theremaining ORFs.

The PCR mixture comprises, for a final volume of 100 μl, 10-50 ng ofgenomic DNA, the N-terminal and C-terminal primers each at 200 nM, thedNTPs each at 250 μM, the 1×PCR buffer (composition of the 10×PCRbuffer: 200 mM Tris-HCl (pH 8.8), 20 mM MgSO₄, 100 mM KCl, 100 mM(NH₄)₂SO₄, 1% TritonX-100 and 1 mg/ml of nuclease-free bovine serumalbumin) and 2.5 U of polymerase.

The amplification is carried out as follows:

Temperature Number of Step (° C.) Time (min.) cycles Denaturation 970.45 25 Hybridization cf. table 1 25 Elongation 72 1/kb DNA 25

The primers used and the PCR conditions given in the table below, inwhich “N. g allelic variant” means that an allelic variant is present inNeisseria gonorrhoeae and “N. m A allelic variant” means that an allelicvariant is present in Neisseria meningitidis serogroup A.

ORF No. (internal ref.) SEQ ID No. 5′ Primer 22 1-2 GCT CTA GAC CAC CATGTC TGA AGA N.g allelic variant: AAA ATT GAA AAT GAG (SEQ ID n^(o) 78)54, 55 41 3-4 GCT CTA GAC CAC CAT GAA ACA CTT ACT CAT CG (SEQ ID n^(o)80) 42-43 5-6 GCT CTA GAC CAC CAT GAA AAA ATC N.g allelic variant: CCTTTT CGT TC (SEQ ID n^(o) 82) 56, 57 47 7-8 GCT CTA GAC CAC CAT GCG AACGAC N.g allelic variant: CCC AAC CTT C (SEQ ID n^(o) 84) 58, 59 55 9-10GCT CTA GAC CAC CAT GAA CAC ACG N.g allelic variant: CAT CAT CGT TTC(SEQ ID n^(o) 86) 60, 61 68 11-12 GCT CTA GAC CAC CAT GCT GAC GTT TATCGG ACT G (SEQ ID n^(o) 88) 71 13-14 GCT CTA GAC CAC CAT GGG CAT CCA TCTCGA CTT C (SEQ ID n^(o) 90) 72 15-16 GCT CTA GAC CAC CAT GAA TAG ACCN.mA. allelic CAA GCA ACC (SEQ ID n^(o) 92) variant: 62, 63 73 17-18 GCTCTA GAC CAC CAT GAT GAA TGT N.g allelic CGA GGC AGA G (SEQ ID n^(o) 94)variant: 64, 65 74 19-20 GCT CTA GAC CAC CAT GAA ATT TTT TCC TGC TCC(SEQ ID n^(o) 96) 98 21-22 GCT CTA GAC CAC CAT GAT TGA ATT TGT CCG AGC(SEQ ID n^(o) 98) 116  23-24 GCT CTA GAC CAC CAT GCA ATA CAG N.g.allelic CAC ACT GGC (SEQ ID n^(o) 100) variant: 66, 67 122  25-26 GCTCTA GAC CAC CAT GGA GCA GTC GGG CAA ATT C (SEQ ID n^(o) 102) 125  27-28GCT CTA GAC CAC CAT GCA AAA CGG CGG GGG AAA G C (SEQ ID n^(o) 104) 128 29-30 GCT CTA GAC CAC CAT GAC ATT GCT N.mA. allelic CAA TCT AAT GAT AATG (SEQ ID n^(o) 106) variant: 68, 69 152  31-32 GCT CTA GAC CAC CAT GAAACA ATC N.g allelic variant: CGC CCG (SEQ ID n^(o) 108) 70, 71 153 33-34 GCT CTA GAC CAC CAT GAA TGT TTA CGG TTT CCC (SEQ ID n^(o) 110)155  35-36 GCT CTA GAC CAC CAT GAT GAG TCA ACA CTC TGC C (SEQ ID n^(o)112) 156  37-38 GCT CTA GAC CAC CAT GCC TTC GAG CAA AAA CTG G (SEQ IDn^(o) 114) 157  39-40 GCT CTA GAC CAC CAT GCA CCT TGG AAA G (SEQ IDn^(o) 116) 158  41-42 GGA CTA GTC CAC CAT GGC TGC CAA N.mA. allelic CCAACG TTA CCG (SEQ ID n^(o) 118) variant: 72, 73 159  43-44 GCT CTA GACCAC CAT GCC GCA AAT N.mA. allelic TAA AAT TCC C (SEQ ID n^(o) 120)variant: 74, 75 161  45-46 GCT CTA GAC CAC CAT GCG CAC GCC GTT TTG TTG(SEQ ID n^(o) 122) 163-1   47-48 GCT CTA GAC CAC CAT GAG AAT AGA GAT CACACC (SEQ ID n^(o) 124) 163-2   49-50 GCT CTA GAC CAC CAT GAT TCA CGT TTCGGC AGT G (SEQ ID n^(o) 126) 167-168 51-52 GCT CTA GAC CAC CAT GAA TTCGAC N.g allelic variant: CGC AAG TAA AAC (SEQ ID n^(o) 128) 76, 77 ORFNo. Hybrid- (internal ization ref.) 3′ Primer Polymerase T° 22 CGG GATCCA GAA ATG GCT GGA TTC Tfu 56° C. GCT ATC AG (SEQID n^(o) 79)(Appligene) 41 CGG GAT CCA ATA CGT AGG ACT TGG Tfu 43° C. GTC (SEQ IDn^(o) 81) (Appligene) 42-43 CGG GAT CCA TTG CGG ATA AAC ATA Tfu 56° C.TTC CGC C (SEQ ID n^(o) 83) (Appligene) 47 CGG GAT CCA GAA CCG GTA GCCTAC Tfu 56° C. GCC GAC (SEQ ID n^(o) 85) (Appligene) 55 CGG GAT CCA GCAACG GCC TGC CGC Pfu Turbo 56° C. TTT AAG (SEQ ID n^(o) 87) (Stratagene)68 CGG GAT CCA CGG CAG AGG CAC GAT Tfu 56° C. TCC (SEQ ID n^(o) 89)(Appligene) 71 CGG GAT CCA CAA AAG TTC CAG AAA Tfu 56° C. ATC TAA CTC(SEQID n^(o) 91) (Appligene) 72 CGG GAT CCA TGC CGC TTG GGG GAG GC PfuTurbo 56° C. (SEQ ID n^(o) 93) (Stratagene) 73 CGG GAT CCA CAG TTT GCCCGA CAT AC Pfu Turbo 56° C. (SEQ ID n^(o) 95) (Stratagene) 74 GAA GATCTA GAA ACT GTA ATT CAA Pfu Turbo 56° C. GTT GAA G (SEQ ID n^(o) 97)(Stratagene) 98 CGG GAT CCA ACC CTG CGA CGA GTT Pfu Turbo 56° C. GCG(SEQ ID n^(o) 99) (Stratagene) 116  CGG GAT CCA GTC CTT TTT CGC ACC TTGPfu Turbo 56° C. AAG (SEQ ID n^(o) 101) (Stratagene) 122  CGG GAT CCAAGC TGT TTG GCG ATT Pfu Turbo 56° C. TCG GTG (SEQ ID n^(o) 103)(Stratagene) 125  CGG GAT CCA GTG CCT GCG CAG CTT Pfu Turbo 56° C. GGAATC (SEQ ID n^(o) 105) (Stratagene) 128  CGG GAT CCA TTC CGC AAA TAC CTGTfu 56° C. TTT CCA ACC (SEQ ID n^(o) 107) (Appligene) 152  CGG GAT CCATAC TTG GGC GCA ACA Pfu Turbo 56° C. TGA C (SEQ ID n^(o) 109)(Stratagene) 153  CGG GAT CCA TTT TTT AGA CGT ATT TTT Tfu 56° C. AGT CG(SEQ ID n^(o) 111) (Appligene) 155  CGG GAT CCA TCC AGT TTT TGC TCG Tfu56° C. AAG GC (SEQ ID n^(o) 113) (Appligene) 156  CGG GAT CCA TCG TTCTTC AAT CTC CAC Tfu 56° C. AAA CG (SEQ ID n^(o) 115) (Appligene) 157 CGG GAT CCA TTC AAT TCG CTT CAA Tfu 56° C. CAA TG (SEQ ID n^(o) 117)(Appligene) 158  GAA GAT CTA AGC CGC GTT CCC TTC Tfu 56° C. CAA AAA ATC(SEQ ID n^(o) 119) (Appligene) 159  CGG GAT CCA AAA ACA ATC TTC CGG Tfu56° C. CAC CC (SEQ ID n^(o) 121) (Appligene) 161  CGG GAT CCA TTG GGCAAC GAC GAA Tfu 56° C. GGC AC (SEQ ID n^(o) 123) (Appligene) 163-1   CGGGAT CCA TGG CTC AAT CCT TTC TGC Pfu Turbo 56° C. (SEQ ID n^(o) 125)(Stratagene) 163-2   CGG GAT CCA ACC TGC TTC ATG GGT Tfu 56° C. GAT TC(SEQ ID n^(o) 127) (Appligene) 167-168 CGG GAT CCA AAT CCC TCT GCC GTATfu 56° C. TTT G (SEQ ID n^(o) 129) (Appligene)

2—Cloning, Transformation and Selection of Recombinants

The cloning vector used is the 6.357 kb vector pCA/Myc-His or pM1070(cf. FIGURE), derived from the plasmid pcDNA 3.1 (Invitrogen).pCA/Myc-His comprises, in particular, the CMV ie1 promoter (bases249-902), intron A of the CMV ie1 gene (Chapman et al., 1991 NucleicAcids Research, 19, 3979-3986), a multiple cloning site (bases1792-1852) with the PmlI, EcoRV, NotI, XbaI, BamHI, KpnI and HindIIIsites, a sequence encoding a polyhistidine and a stop codon (bases1908-1928), a bgh 3′ termination sequence (bases 1853-2197) and theampicillin resistance gene for selecting the recombinant clones in E.coli.

After purification (GeneClean Bio101 kit), the PCR amplificationproducts are digested for 2 hours at 37° C. with the appropriate enzymes(XbaI-BamHI, XbaI-BglII or SpeI-BglII), in a final reaction volume of 20μl. The digestion products are then ligated with the vector pCA/Myc-His,digested beforehand with XbaI and BamHI, according to the “Rapid DNALigation Kit” protocol (Boehringer Mannheim). 15 μl of the ligation isused to transform 100 μl of competent E. coli XLI-blue cells (Novagen).The cells are incubated for 30 minutes in ice, 30 seconds at 42° C. and2 minutes in ice. Then, 500 μl of LB medium without antibiotics areadded, and the mixture is incubated for 1 hour at 37° C. Next, 50 and550 μl of the culture are plated out on plates containing LB medium plusampicillin (50 μg/ml final concentration), and incubated overnight at37° C.

The following day, 36 colonies are placed in culture in 2 ml of LB plusampicillin (50 μg/ml) and incubated overnight at 37° C.

The following day, the plasmid DNA is extracted according to the Qiagenmini-prep protocol (Qiagen) and the recombinants are identified byenzymatic restriction followed by agarose gel electrophoresis. Thecloning junctions are then verified by sequencing.

EXAMPLE II Evaluation of the Protective Activity of the ORFs of theInvention

A. Preparation of the DNA Intended for the Immunization Experiments:

An isolated colony of a recombinant clone is used to inoculate apreculture in LB medium+ampicillin, and 5 ml of this preculturerepresents the inoculum of a 2.5 litre culture in LB medium+ampicillin.The purification protocol for preparing the plasmid DNA is thatdescribed in the EndoFree Giga Kit (Qiagen). The purified DNA is elutedfrom the purification column with a 10 mM Tris-HCl, 1 mM EDTA buffer, pH8, and stored at −20° C. Before injection, the purified recombinantplasmid is diluted to 100 μg/ml with water (of injectable preparationquality) and the NaCl concentration is brought to 150 mM.

B. Production of a Specific Polyclonal Serum:

B.1. Hyperimmunization in an Animal Model:

The animal model used is the mouse or the rabbit. The route ofadministration of the injected DNA is the intramuscular or intradermalroute. The recombinant plasmids to be injected are optionally applied tobeads if they are injected into animals using a gene gun apparatus(BioRad). The immunization protocol follows a scheme comprising twoinjections, 3 weeks apart.

B.2. Analysis of the Bactericidal Activity of the Antibodies Induced:

Ten days after the final injection, the animals are bled and the seraare analyzed using the bactericidal activity assay according to theprotocol of Danve et al., Vaccine (1993) 11 (12):1214. Briefly, the seraare incubated at various dilutions (2-fold) in the presence of rabbitcomplement and of meningococci cultured in the presence or absence of aniron-chelating agent. The bactericidal titre of a serum represents theinverse of the dilution of this antiserum for which 50% of the bacteriaare lysed.

It is considered that the antiserum is not bactericidal when its titreis lower than 4 against the homologous strain.

When the bactericidal titre corresponds to a 4-fold seroconversionagainst the homologous strain, the bactericidal activity of theantiserum is analyzed against other Neisseria strains in order tomeasure the extent of the cross-reactivity of the antiserum of interest.

EXAMPLE III Production of Purified Recombinant Proteins

1. Recombinant Production of Proteins

a. Preparation of Transformants:

The PCR product obtained is then digested at 37° C. for two hours withrestriction enzymes, in 20 μl of reaction volume. The digestion productis ligated into a plasmid pET28a (Novagen) which is cleaved in a similarway and which is dephosphorylated, before ligation, by treating withcalf intestine alkaline phosphatase. The fusion gene constructed in thisway allows the one-step affinity purification of the resulting fusionprotein, due to the presence of histidine residues at the N-terminal endof the fusion protein, which are encoded by this vector.

The ligation reaction (20 μl) is carried out at 14° C. overnight, beforetransforming 100 μl of fresh competent E. coli XL1-blue cells (Novagen).The cells are incubated on ice for two hours, and then subjected to aheat shock at 42° C. for 30 seconds, before being returned to the icefor 90 seconds. The samples are then added to 1 ml of LB broth withoutselection, and cultured at 37° C. for two hours. The cells are thenplated out on LB agar medium supplemented with kanamycin (50 μg/ml finalconcentration) at a 10× dilution, and are incubated overnight at 37° C.The following day, 50 colonies are subcultured on secondary plates andare incubated at 37° C. overnight.

b. Production of the Protein:

The stored transformants (10 μl) are plated out onto selection platesand cultured overnight at 37° C. A few cells are harvested from theplate and used as an inoculum for an overnight starter culture (3 ml) at37° C. The following day, a sample (time T=0) is taken and centrifugedat 14 000 rpm for 3 minutes. The starter culture is then used toinoculate an LB medium containing kanamycin (100 μg/ml) at a dilution of1:50 (starting optical density OD₆₀₀=0.05-0.1). The cells are culturedto an OD₆₀₀ of 1.0, a sample is taken for SDS-PAGE (pre-inductionsample) and the remaining culture is induced with 1 mM of IPTG. Thecultures are cultured for four hours and samples are taken every hour.The culture is centrifuged at 600 g for 20 minutes at 4° C. Thesupernatant is discarded and the pellets are resuspended in 50 mM ofTris-HCl (pH: 8.0), mM EDTA, and recentrifuged. The supernatant isdiscarded and the cells are stored at −70° C.

2. Protein Purification

The pellets obtained from one litre of culture prepared according toExample I.4 above are dried and resuspended in 20 ml of 20 mM Tris-HCl(pH 8.0), 0.5 M NaCl, 5 mM imidazole, cooled in ice. Lysozyme is addedat a concentration of 0.1 mg/ml, and the suspension is homogenized usinga high-speed homogenizer (Turrax), then treated with a sonicator(Sonifier 450, Branson). Benzonase (Merck) is used at a finalconcentration of 1 U/ml in order to eliminate the DNA. The suspension iscentrifuged at 40 000 g for 20 minutes and the supernatant is filteredthrough a 0.45 μm membrane. The supernatant is loaded onto an IMACcolumn (12 ml of resin) which has been prepared by immobilizing Ni⁺⁺cations according to the manufacturer's recommendations (Pharmacia). Thecolumn is washed with 10 column volumes of 20 mM Tris-HCl (pH 8.0), 0.5M NaCl, 60 mM imidazole. The recombinant protein is eluted with sixvolumes of 20 mM Tris-HCl (pH: 7.9), 0.5 M NaCl, 500 mM imidazole, 0.1%Zwittergent 3-14.

The elution profile is controlled by measuring the absorbence of thefractions at an optical density of 280 nm. An aliquot fraction isanalyzed on an SDS-PAGE gel and stained with Coomassie blue (PhastSystem—Pharmacia), and the fractions corresponding to the protein peakare then pooled and concentrated. In order to eliminate the elutionbuffer, the fraction is passed over a G24 Sephadex column (Pharmacia)and equilibrated in PBS buffer (pH: 7.4). The protein solution issterilized by filtration through a 0.45 μm membrane, and the proteinconcentration is determined using the BCA micromethod (Pierce). Theprotein solution is stored at −70° C.

EXAMPLE IV Production of Monospecific Polyclonal Antibodies

1. Rabbit Hyperimmune Antiserum

100 μg (in total) of the polypeptide purified in Example III, in thepresence of complete Freund's adjuvant in a total volume ofapproximately 2 ml, are injected into New Zealand rabbits, bothsubcutaneously and intravenously. 21 and 42 days after the initialinjection, the booster doses, which are identical to the initial doses,are administered in the same way, with the exception that incompleteFreund's adjuvant is used. 15 days after the final injection, theanimal's serum is recovered, decomplemented and filtered through a 0.45μm membrane.

2. Mouse Hyperimmune Ascites Fluid

10-50 μg of the purified fusion polypeptide obtained in Example II, inthe presence of complete Freund's adjuvant, in a volume of approximately200 μl, are injected subcutaneously into 10 mice. 7 and 14 days afterthe initial injection, booster doses, which are identical to the initialdoses, are administered in the same way, with the exception thatincomplete Freund's adjuvant is used. 21 and 28 days after the initialinjection, the mice receive 50 μg of the antigen alone,intraperitoneally. On the 21st day, the mice are also injectedintraperitoneally with 180/TG CM26684 sarcoma cells (Lennette & Schmidt,Diagnostic procedures for viral, rickettsial, and chlamydial infections,(1979) 5th Ed. Washington D.C., American Public Health Association). Theascites fluids are harvested 10 to 13 days after the first injection.

EXAMPLE V Purification of the Polypeptides of the Invention byImmunoaffinity

1. Purification of Specific IgG

An immune serum as prepared in Example IV is applied to a Fast FlowSepharose 4 protein A column (Pharmacia) equilibrated with 100 mMTris-HCl (pH: 8.0). The resin is washed by applying 10 column volumes of100 mM Tris-HCl and 10 volumes of 10 mM Tris-HCl (pH: 8.0) to thecolumn. The IgGs are eluted with a 0.1 M glycine buffer (pH: 3.0) andare collected by 5 ml fraction, to which 0.25 ml of 1 M Tris-HCl (pH:8.0) are added. The optical density of the eluate is measured at 280 nmand the fractions containing the IgGs are pooled and, if necessary,stored at −70° C.

2. Column Preparation

A suitable amount of CNBr-activated Sepharose 4B gel (1 g of dried gelproviding approximately 3.5 ml of hydrated gel, and the capacity of thegel ranging from 5 to 10 mg of coupled IgG per ml of gel) manufacturedby Pharmacia (17-0430-01) is suspended in 1 mM HCl buffer and washed,using a Buchner funnel, by adding small amounts of 1 mM HCl buffer. Thetotal volume of the buffer is 200 ml per gram of gel.

The purified IgGs are dialysed for four hours at 20±5° C. against 5volumes of 500 mM PBS buffer (pH: 7.5). Then, they are diluted in 500 mMof PBS (pH: 7.5) for a final concentration of 3 mg/ml.

The IgGs are incubated with the gel overnight at 5±3° C., with stirring.The gel is packed into a chromatography column and washed with 2 columnvolumes of 500 mM phosphate buffer (pH: 7.5) and then one volume of 50mM NaCl sodium buffer (pH: 7.5). The gel is then transferred to a tube,then incubated with 100 mM of ethanolamine (pH: 7.5) for 4 hours at roomtemperature with stirring, and then washed twice with two column volumesof PBS. The gel is then stored in PBS merthiolate at 1/10 000. Theamount of IgG coupled to the gel is determined by measuring the opticaldensity at 280 nm of the IgG solution and of the direct eluate.

3. Adsorption and Elution of the Antigen

A solution of antigen in 50 mM Tris-HCl (pH: 8.0), 2 mM EDTA, forexample the supernatant obtained in Example III.2 after treatment withBenzonase, centrifugation and filtration through a 0.45 μm membrane, isapplied to a column equilibrated with 50 mM Tris-HCl (pH: 8.0), 2 mMEDTA, at a flow rate of approximately 10 ml/hour. Then, the column iswashed with 20 volumes of 50 mM Tris-HCl (pH: 8.0), 2 mM EDTA.Alternatively, batch adsorption can be carried out, in which the mixtureis left overnight at 5±3° C., with stirring.

The gel is washed with 2 to 6 volumes of 10 mM PBS buffer (pH: 6.8). Theantigen is eluted with a 100 mM glycine buffer (pH: 2.5). The eluate iscollected in 3 ml fractions, to which 150 μl of 1 mM PBS buffer (pH:8.0) are added. The optical density is measured at 280 nm for eachfraction; those containing the antigen are recovered and stored at −20°C.

Fragments of the Genome of N. meningitidis Z2491 Described in PatentApplication WO 98/02547

(2) INFORMATION FOR SEQ ID NO: 130:

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 243 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130:

GATCAGACCC ATTTTCAGCG CACCGTAAGC GCGGATTTTC TCGAATTTTT CCAAAGCTGC 60GGCATCGTTG TTGATGTCGT CTTGCAACTC TTTGCCCGTG TAGCCCAAGT CGGCGGCATT 120CAGGAAAACG GTCGGAATGC CCGCGTTGAT GAGCGTGGCT TTCAAACGGC CTATATTCGG 180CACATCAATT TCATCGACCA AATTGCCGGT TGGGAACATA CTGCCTTCGC CGTCGGCTGG 240ATC 243(2) INFORMATION FOR SEQ ID NO: 131:

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 120 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131:

CGGTCAGAAA CAGGCAAGGT AATGAAAATG CCTGAGGCAC GGACTGTGCT GCGAACGAAA 60ACTCCTTACC GAAGTCTTCT ATACCCAGGC TCAATAGCCG CTCAAGGAGA GAGCTATCAT 120(2) INFORMATION FOR SEQ ID NO: 132:

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 120 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 132:

CGGTCAGAAA CAGGCAAGGT AATGAAAATG CCTGAGGCAC GGACTGTGCT GCGAACGAAA 60ACTCCTTACC GAAGTCTTCT ATACCCAGGC TCAATAGCCG CTCAAGGAGA GAGCTATCAT 120(2) INFORMATION FOR SEQ ID NO: 133

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 269 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133:

CGGAGCATAA AATCGTTATT AAAGATAATG GTATAGGAAC GAGCTTCGAT GAAATCAATG 60ATTTTTATTT GAGAATCGGT CGGAACAGAA GGGAAGAAAA ACAAGCCTCC CCGTGCGGAA 120GAATTCCAAC GGGTAAAAAA GGCCTTGGTA AATTGGCATT ATTCGGGCTT GGCAACAAAA 180TTGAAATTTC TACTATCCAG GGAAACGAAA GGGTTACTTT TACTTTGGAT TATGCAGAGA 240TTCGAAGAAG CAAGGGTATT TATCAACCG 269(2) INFORMATION FOR SEQ ID NO. 134:

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 207 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134:

CGGGTCGCTT TATTTTGTGC AGGCATTATT TTTCATTTTT GGCTTGACAG TTTGGAAATA 60TTGTGTATCG GGGGGGGGTA TTTGCTGACG TAAAAAACTA TAAACGCCGC GCAAAATATG 120GCTGACTATA TTATTGACTT TGATTTTGTC CTGCGCGGTG ATGGATAAAA TCGCCAGCGA 180TAAAGAATTT GCGAGAACCT GATGCCG 207(2) INFORMATION FOR SEQ ID NO. 135:

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 224 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135:

CGGCAACGAT TTGAGCTATC GCGGTTACGA CATTCTGGAT TTGGCACAAA AATGCGAGTT 60TGAAGAAGTC GCCCACCTGC TGATTCACGG CCATCTGCCC AACAAATTCG AGCTGGCCGC 120TTATAAAACC AAGCTCAAAT CCATGCGCGG CCTGCCTATC CGTGTGATTA AAGTTTTGGA 180AAGCCTGCCT GCACATACCC ATCCGATGGA CGTAATGCGT ACCG 224(2) INFORMATION FOR SEQ ID NO: 136:

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 273 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136:

AATTTCCACC TATGCCCTAC GCAGCGATTA TCCGTGGTTT ACCCAAAGGG TGATTATGGC 60AAAAGCGCGG GGTTGAGCGA CCGCCTTTTG TTGCCGGCGT TCAAACGGGT TTTGATAGGA 120AATGCAGGCA CGAAGCCTCG GCTGATTGTG ATGCACCTGA TGGGTTCGCA CAGTGATTTT 180TGCACACGTT TGGATAAGGA TGCGCGGCGG TTTCAGTATC AAACTGAAAA AATATCCTGC 240TATGTTTCCA TCAATCGCGC AAACCGATAA ATT 273(2) INFORMATION FOR SEQ ID NO: 137:

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 270 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137:

AATTCTTCCG CACGGGGAGG CTTGTTTTTC TTCCCTTCTG TTCCGACCGA TTCTCAAATA 60AAAATCATTG ATTTCATCGA AGTTCATTCC TATACCATTA TCTTTAATAA CGATTTTATG 120CTCCGGTTTA TCGAATAACC TAACTTCCAC TTCCGTAGCA CATGCATCGT AGGCATTCGC 180TATCAACTCG GCAATCGCAG GAACAGTGTG CGAATACAAT CTTTACACCC AAATGTTCGA 240TTACGGTTGG CTCGAAACTC AATTTCAATT 270(2) INFORMATION FOR SEQ ID NO: 138:

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 267 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 138:

AATTATGAAC ACACGCATCA TCGTTTCGGC TGCGTTCGTT GCGTTGGCAT TAGCAGGTTG 60CGGCTCAATC AATAATGTAA CCGTTTCCGA CCAGAAACTT CAGGAACGTG CCGCGTTTGC 120CTTGGGCGTC ACCAATGCCG TAAAAATCAG CAACCGCAGC AATGAAGGCA TACGCATCAA 180CTTTACCGCA ACTGTGGGTA AGCGCGTGAC CAATGCTATG TTACCAGTGT AATCAGCACA 240ATCGGCGTTA CCACTTCCGA TGCAATT 267(2) INFORMATION FOR SEQ ID NO: 139:

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 308 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139:

AATTTGTTGG GCAGATGGCC GTGAATCAGC AGGTGGGCGA CTTCTTCAAA CTCGCATTTT 60TGTGCCAAAT CCAGAATGTC GTAACCGCGA TACGTCAAAT CGTTGCCGGT ACGCAACGGT 120ACACAAAGCG GTATTACCGG CCGCAACGCC AGAAAGCGCA ACGGATTTTT AGGTTTGAGG 180GTCGGGGTTT GAGTAGTTTC AGTCATGGTA TTTCTCCTTT GTGTTTTTAT GGCTTTCGGG 240TTTTCAGACG ACCGATGCGG ATTTGTTGAA AGGCAGTCTG AAAGCGGTAA ATCATTTTTG 300AAACAATT 308(2) INFORMATION FOR SEQ ID NO: 140:

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 286 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140:

AATTCGGAGG AGCAGTACCG CCAAGCGTTG CTCGCCTATT CCGGCGGTGA TAAAACAGAC 60GAGGGTATCC GCCTGATGCA ACAGAGCGAT TACGGCAACT TGTCCTACCA CATCCGTAAT 120AAAAACATGC TTTTCATTTT TTCGGCAAGC AATGACGCAC AAGCTCAGCC CAACACAACT 180GACCCTATTG CCATTTTATG AAAAAGACGC TCAAAAAGGC ATTATCACAG TTGCAGGCGT 240AGACCGCAGT GGAGAAAAGT TCAATGGCTC CAACCATTGC GGAATT 286(2) INFORMATION FOR SEQ ID NO: 141:

(i) SEQUENCE CHARACTERISTICS:

-   -   (A) LENGTH: 316 base pairs    -   (B) TYPE: nucleotide    -   (C) STRANDEDNESS: single    -   (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iv) ANTISENS: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141:

AATTTGTCGG CAATCTTCCC GGGTCGCTTT ATTTTGTGCA GGCATTATTT TTCATTTTTG 60GCTTGACAGT TTGGAGATAT TGTGTATCGG GGGGGGGTAT TTGCTGACGT AAAAAACTAT 120AAACGCCGCA GCAAAATATG GCTGACTATA TTATTGACTT TGATTTTGTC CTGCGCGGTG 180ATGGATAAAA TCGCCAGCGA TAAAGATTTG CGAGAACCTG ATGCCGGCCT GTTGTTGAAT 240ATTTTCGACC TGTAATTACG ATTTGGCTTC CGCGCCGGCA CAATATGCCG CCAAGCGGCG 300CCCACATTTT GGAAGC 316

1. An isolated polypeptide comprising the amino acid sequence SEQ ID NO:8 or SEQ ID NO:
 53. 2. The polypeptide according to claim 1, whereinsaid polypeptide consists of an amino acid sequence of SEQ ID NO:
 53. 3.The polypeptide according to claim 1, wherein said polypeptide consistsof an amino acid sequence of SEQ ID NO:
 8. 4. A composition comprising apharmaceutically acceptable vehicle in combination with the isolatedpolypeptide according to claim
 1. 5. The composition according to claim4, wherein said polypeptide consists of an amino acid sequence of SEQ IDNO:
 53. 6. The composition according to claim 4, wherein saidpolypeptide consists of an amino acid sequence of SEQ ID NO: 8.